The Polymerase Chain Reaction (PCR) has proven to be a versatile and powerful technique for amplifying nucleic acids. The PCR utilizes the ability of natural or recombinant DNA polymerases to reproduce a target nucleic acid to high levels. Theoretically, this procedure is capable of producing logarithmic reproductions, (amplification) of a single copy of DNA. However, the sensitivity of the PCR process is compromised by a number of factors during the amplification process, resulting in a significant loss of sensitivity. One of the major problems is the development of a non-specific product during the reaction, commonly known as “primer-dimers”. When these products form, they result in the removal of both primers and deoxyribonucleoside triphosphates (dNTPs) from the reaction, thereby reducing the level of amplification of the desired target and concurrently reducing sensitivity of the reaction.
In order to avoid these deficiencies, the “hot start” PCR technique was developed. A hot start reaction involves preventing the PCR reaction from occurring at lower, non-specific temperatures before temperature cycling is initiated and amplification ensues. Hot-start reaction techniques have been based on inactivation of the polymerase at the lower temperature, or alternatively withholding a critical component of the PCR reaction, such as polymerase, nucleoside triphosphates, or Mg2+.
In the initial hot start technique the reaction mixture was heated above the annealing Tm of the primers prior to addition of a critical component of the reaction. However, this method is cumbersome, prone to contamination and is not amenable to high throughput application since multiple additions must be performed. It also involves the use of a mineral oil overlay since addition of reagents to reaction must be performed at elevated temperatures. Additionally, once reaction begins dimer inhibition no longer occurs.
A similar method to the one described above is the wax barrier method where mineral oil is replaced with a wax that liquefies at high temperatures, described in Chou et al., Nucleic Acids Res 20 (7), p. 1717 (1992). The reaction mixture is heated and cooled prior to addition of a critical component of the reaction causing the wax to harden forming a barrier. A limiting component is added on top of the wax above reaction mix, and when temperature cycling is initiated, the wax melts allowing the denser aqueous limiting reagent to sink through the liquid wax forming complete reaction mixture and amplification ensues. Unfortunately, this technique requires cumbersome heating-cooling-addition of limiting reagent. Additionally, wax must be added to each well as a solid pellet and thus the method is very low throughput and cannot be automated. Certain waxes have higher degrees of opacity so the use of fluorescence detection for real-time PCR can be limited.
Another hot start technique involves conjugating the polymerase with an antibody (Ab) or mixture of antibodies directed against the polymerase protein as described in U.S. Pat. No. 5,338,671. The Ab inactivates the polymerase at low temperatures and when the reaction is heated the Ab is irreversibly inactivated due to denaturation. Ab inactivation allows polymerase to become active during subsequent annealing and extension steps allowing PCR to occur. This technique is also subject to several limitations including the fact that the Ab is expensive and is specific only to a single polymerase. Thus, a new Ab must be isolated to react with each type of polymerase. This methodology also cannot be used with reverse transcriptase (RT) because elevated temperature sufficient to inactivate the Ab also inactivates the RT.
Another method involves chemical modification of polymerase enzyme with a chemical moiety such as a cyclic anhydride that attaches to lysine as disclosed in U.S. Pat. No. 6,183,998. Derivatization inactivates the polymerase which when incubated at an elevated temperature in the presence of a temperature-sensitive buffer, such as Tris, results in a significant pH decrease at 95° C. The acid conditions resulting at this elevated temperature reverse the chemical derivitization and activates the enzyme. The drawback of this technique includes the requirement of long-term incubation, generally greater than 10 minutes at denaturing temperature. Acid sensitive fluorophore detection chemistries can be adversely affected by the resulting pH changes. This methodology also cannot be used with RT because elevated temperature sufficient to inactivate the anhydride also inactivates RT. Moreover, reversal of chemical derivative is not efficient and the full activity of enzyme is not recovered necessitating increasing enzyme concentrations for many applications.
One hot start method that sequesters magnesium involves the addition of phosphoric acid to buffer causing room temperature precipitation of magnesium ions that are required for PCR. See, Barnes et al., Mol Cell Probes 16 (3), p. 167 (2002) and U.S. Pat. No. 6,403,341. Incubation of the reaction mixture at 95° C. resolubilizes the magnesium precipitate and magnesium ions will stay in solution at elevated reaction temperatures of PCR. In that method, efficient precipitation of the magnesium ion is dependent on the use of a special buffer. Also inhibition of PCR may not be complete because not all of the magnesium ion precipitates. As above, acid conditions may adversely affect sensitive fluorophores or other moieties such as isobases and this methodology also cannot be used with RT because elevated temperature sufficient to inactivate the anhydride also inactivates the RT
Still another hot start technique is based on the use aptamers, polypeptides or single-stranded nucleic acids that are selected to be specific for a particular polymerase. The aptamer method involves selection and amplification of structured nucleic acids, using the SELEX technique, that specifically bind to and inhibit the polymerase. These techniques are described in U.S. Pat. Nos. 5,693,502, 5,763,173, 5,874,557, 6,020,130, and 6,183,967 and Dang et al. J Mol Biol 264 (2), p. 268 (1996). Selection is done at low temperatures and incubation of the aptamer at elevated temperatures denatures the structural elements required for specific inhibition. Denatured aptamer can no longer inhibit polymerase activity allowing PCR. However, the aptamer is specific only to a single polymerase or closely related polymerases. Thus, a new aptamer is required to react with each type or family of polymerase. Also temperature optima of aptamers may not be easily predicted or controlled and inhibitory activity of the aptamer may not be fully reversed at a given temperature necessitating precise optimization of aptamer/enzyme concentration and reaction conditions.
Another technique describes reversible solid-phase attachment of polymerase HSA fusion protein to achieve hot start. Nilsson et al., BioTechniques 22 (4), p. 744 (1997).
Accordingly, there remains a need for a simplified method and reagents for inhibiting or preventing non-specific nucleic acid extension and/or amplification in a PCR reaction.